Gene Synthesis
Handbook
Fourth Edition

A Guide to Gene Synthesis Design and Applications

Custom Gene Synthesis
Take Control of Your Research with GenScript

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Every Step

Gene Expertise from
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Industry-leading OptimumGene
Codon Optimization Technology

Table of Contents
Introduction
Definition and history
Types of synthetic products
Significance357Applications
Core applications
Generating nucleic acid-based reagents
Codon optimization
Mutagenesis

13
13
15
17

Broad applications
Synthetic biology
Translational research and medicine
Agriculture and environment

20
20
28
31

Technology
Sequence design and applications
Synthesis
Assembly
Sequence verification and error correction
Downstream application preparation

34
37
40
45
46

Perspectives
Biosafety
Future52
52

Resources
Tools
Design considerations

1

55
56

Chapter One
Introduction

Definition and History

Gene synthesis refers to the base-by-base process of chemically synthesizing a strandof DNA. Unlike traditional methods of DNA replication, gene synthesis does not require
a template strand. Rather, gene synthesis involves the step-wise addition of nucleotides
to a single-stranded nucleic acid molecule, which then serves as a template for thesynthesis of the complementary strand. Sequences with varying lengths can be synthesized as stand-alone genes or building blocks corresponding to a larger gene, which
are then enzymatically assembled together. The gene synthesis technology is a powerful and rapidly advancing tool, which has significantly affected all areas of biology withexciting and far-reaching implications.
The history of gene synthesis began in 1955, when Sir Alexander Todd published achemical method for creating a phosphate link between two thymidine nucleotides;
effectively describing the first artificial synthesis of a DNA molecule1. Based on thismethod, in 1970 Gobind Khorana’s group reported the successful synthesis of an entire
gene, the 77 kilo base (kb) pairs gene for a yeast tRNA, in the span of five years2. Development of phosphoramididite DNA synthesis by Caruthers and Matteucci in 1983 wasfast followed by commercial availability of automated oligonucleotide synthesizer
machines in 1989. Concentrated efforts in the 1990s, lead to the development of
improved assembly methods that could enable the synthesis of longer genes. A
decade later, the synthesis of an entire viral genome3 in 2003 paved the way for thelaunch of the Synthetic Yeast Genome Project (Sc2.0) as the world’s first synthetic
eukaryotic genome project. This global collaborative effort aimed to create a novel,
rationalized version of the genome of the yeast species Saccharomyces cerevisiae,
comprised of 16 designer synthetic chromosomes encompassing 12 million bp of DNA.
Scientists aim to build upon the success of the Sc2.0 project to tackle the challenges
faced in synthetic biology, with the end goal of revolutionizing applications in cellbiology and medicine.

3Timeline of Gene Synthesis Technology Development
1950
DNA polymerase
isolated from E. coli.
Arthur Kornberg
wins 1957 Nobel
Prize.
1967
DNA ligase isisolated andcharacterized by
five independent
laboratories.

1970
First synthesis ofan entire gene, a
77 bp yeast tRNA,
by Gobind
Khorana's group.

1955
First DNA moleculesynthesized when
two thymidine
nucleosides arejoined by aphoshate link. Sir
Alexander Todd
wins 1957 Nobel
Prize.

2004
Microchip- andmicrofluidics-based
methods forhigh-throughput
gene synthesis are
first published.

1983
Polymerase
chain reaction(PCR) isinvented. Kary
Mullis wins 1993
Nobel Prize.
1990s
Improvedassembly
methods allow
synthesis ofincreasingly longgenes
(See page 25).

1989
First automatedoligonucleotide
synthesizer
machines becomecommerciallyavailable.

1983
Caruthers andMatteucci invent
phosphoramidite
DNA synthesis.

2011
Sc2.0 project
launched to build
the first synthetic
eukaryotic
genome (Seepage 16).

2008
First
completesynthesis ofa bacterial
genome.

2003
Firstsynthesisof an entire
viral genome,that of phiX174
bacteriophage.

2014
Expandedapplications
beyondbiomedical
sciences, suchas data science.

4

In the past 60 years, methods for de novo chemical synthesis of DNA have been extensively refined. Continual decreases in cost, advancements in assembly techniques anderror reduction, and improvements in high throughput capabilities through automationand platform integration is rapidly making de novo gene synthesis the preferred
method for molecular biology and bioengineering. These advancements along withimproved methods for sequencing and amplification have now made it possible tosynthesize genes larger than 1 kb in just a few days. Moreover, synthesis of entire
genomes and cost-effective outsourcing to commercial providers is rapidly changing
how biological research is being conducted4. Now, questions about the feasibility of
biological research have shifted from technological limitations towards envisioning
new paradigms for unravelling the mysteries of life and utilizing them for societal
benefits.

Evolution of de novo-Synthesized DNA Over Time107
DNA Length (bp)GenomeGene

Oligonucleotides

1
1960Time (year)

2020* Adapted from Carr et al. 2009

Types of Synthetic Products
Gene synthesis technology can be used to generate a wide variety of nucleic acidproducts for diverse research applications. These products range from naturalsequences to completely de novo nucleic acids in the form of DNA, RNA, or chimeric
DNA-RNA backbones. The length of a synthetic nucleic acid can range from a few basepairs to several kb if assembled in a standard molecular biology laboratory, or over 10
kb if synthesized by specialized gene synthesis providers.

5Dexoyribonucleic Acid (DNA)-Based Product
DNA Oligonucleotides, synthesized as single stranded, short (18-22 nt) fragments,
are commonly used as primers for sequence amplification, sequencing, and mutagenesis. They also can serve as probes in hybridization experiments. Oligo nucleotide libraries or pools can also be used for screening purposes.
Complementary DNA (cDNA), corresponding to the open reading frame (ORF) of agiven gene, can be synthesized for a variety of gene expression studies. Synthesized
genes can encode either naturally occurring transcripts or custom-designed
variants, including point mutations and reporter tags. In addition, synthetic cDNAs
can also be modified for improved gene expression through codon optimization.
Genomic DNA can be synthesized for the study of gene regulatory elements, mRNA
processing, or to create artificial genes and genomes. Synthetic genomic DNA canbe studied in vitro through the creation of transgenic animal lines or transfection ofcell lines in culture. Conventional gene targeting of embryonic stem cells with
disruption cassettes inserted through restriction enzymes5 or Cre-loxP system6 canbe made simpler, more rapidly, and less expensively by using synthetic DNA in
combination with newly developed methods for genome editing such as CRISPR/Cas-9 nucleases, Zinc-Finger Nucleases (ZFNs) or Transcription Activator-Like
Nucleases (TALENs)7.
Extracellular DNA (eDNA) is a component of the organic matter pool in the environment and play important roles in innate immune responses8, thrombosis9, cancer
metastasis10, and biofilm formation11 among other biological functions. Gene
Synthesis technology can fabricate any DNA sequence to further elucidate its roleoutside the cell.

Ribonucleic Acid (RNA)-Based Products
RNA Oligonucleotides are used as primers for hybridization experiments and ascomponents in CRISPR genome editing. Oligo libraries or pools of oligonucleotides
can also be used for targeting and editing studies.
Interfering RNA can be synthesized in a variety of formats to suppress gene expression. Small interfering RNA (siRNA) sequences of 21-25 bp can block gene expression by targeting complementary sequences within mRNA for degradation orsequestration. siRNAs synthesized as RNA oligonucleotides can anneal to form RNA
duplexes, but these molecules are short-lived and often difficult to deliver into cells.
The alternative is vector-based DNA constructs that encode short hairpin RNA

6

(shRNA) molecules which are easier to deliver through viral transduction, plasmidtransfection or electroporation. This will allow the stable expression of shRNA under
the control of inducible or tissue-specific promoters. Upon transcription, shRNA
sequences form a stem-and-loop structure that is cleaved to produce the biologically active siRNA. The Targeted silencing of gene expression through siRNAs enables
the study of genes whose complete knock-out would be otherwise lethal.
Vector-based siRNAs delivered through lentiviruses can also be used cost-effectively
to generate stable cell lines or transgenic animals12.

SignificanceGene synthesis technology has revolutionized biology research and is starting to
impact areas outside of life sciences. The scope of the applications that can utilize genesynthesis has expanded beyond traditional laboratory settings, even reachingdisciplines like data science. Major advantages of gene synthesis technology can be
described in two categories: overcoming limitations of previous technologies to simplify the creation of nucleic acid-based tools, and offering novel opportunities for creating
the foundation of the field of synthetic biology and expanding its applications.

Simplifying Tools Development
Groundbreaking discoveries underlying the molecular cloning technology in the past
several decades have evolved this valuable technology into one of the most powerful
resources in science. However, despite recent improvements in recombinant tools,
synthetically obtaining a clone of interest is hardly a fool-proof endeavor. Molecularcloning strategies have intrinsic limitations, which can not only reduce the pace andincrease the cost of scientific discoveries, but also hamper the expansion of science asa whole. This is where gene synthesis can help complement and overcome theshortcomings of molecular cloning.

7

Gene Synthesis vs Molecular Cloning
Technique

FeatureTraditional molecular cloning

Limited to sequences that appear in nature
Codon optimization is not feasible
Time- and resource-intensive for end-user

Gene synthesis of DNA
fragments

Customizable sequence; no template required
Error-prone; yields mixed pools of variants
Time consuming to verify synthesized sequences
Lower cost for high-throughput screens

Gene synthesis with subcloning
into a vector

Customizable sequence; no template required
Uniformly accurate sequences from a clone
Easy to verify synthesized sequences
Fast and cost-effective

The following summarizes major issues with molecular cloning strategies:
RNA yield and stability: Starting material often needs to be extracted from a livingorganism. The unstable nature of RNA, inefficiency in extraction methods, and low
mRNA copy number can significantly affect the efficiency of this initial step. A projectrisks failure if the starting material cannot be obtained.
Polymerase Chain Reaction (PCR) program optimization: Robust PCR is key inboth sequence amplification and PCR-mediated site-directed mutagenesis.
Because of the unique sequence characteristics of each gene and potential
complexities that may arise from site-directed mutagenesis, PCR optimization can beboth time-consuming and expensive.
DNA yield: In the process of cloning, DNA purification is required at several steps. At
each step, the percentage of DNA recovery can vary from as high as 95% to as low as
60%. Low recovery rates and the presence of contaminants from the purification
procedure can result in the loss of DNA.
Ligation efficiency: The ligation reaction joining the gene insert and vector is a key
step in the cloning process. Several factors, such as the insert to vector ratio, salt
concentration, phosphorylation or de-phosphorylation steps, and solvent contamination can reduce the efficiency of the cloning process.
8

Mutations: The multi-step process of molecular cloning, specifically insert andvector amplification, increases the chances of introducing unwanted mutations.
While the use of proof-reading polymerases can reduce this possibility, their specificreaction conditions, such as sustained high temperature, can in turn lead to DNA
damage and mutations.
Protein quality and yield: The goal of molecular cloning is to obtain a high quality,
functional protein at the desired amount. However, two factors can significantly
affect this outcome. First, toxic genes introduced during the vector propagation step
can increase the survival of mutation-containing transformants while leading to thedeath of correct transformants. This can ultimately affect the expression of a lesspotent version of the target protein. Secondly, constructs designed for heterologous
expression often result in low protein yields due to several reasons, such as
variations in codon usage across species. These factors can ultimately undermine
the main objective of the cloning.
Engineered sequences: The requirement for a naturally-occurring template limits
the application of traditional molecular cloning where novel or customizable
sequences need to be engineered. Although modifying sequences can be accomplished using a cloning strategy, complexities and time required, significantly
hinders its application for novel scientific endeavors.

Express Cloning Services from GenScript eliminate the hands-ontime and uncertainty of traditional molecular cloning techniques. Simply
enter your desired DNA sequence online, select your vector, and receive
your sequence-perfect clone with guaranteed on-time delivery.

The intrinsic characteristics of gene synthesis technology have made it the preferredalternative for efficiently developing a variety of DNA tools. These features include:
Template-independent: With this technology, any existing or imagined sequence
regardless of its complexity or novelty can be synthesized de novo.
Limited-steps process: Compared to steps involved in molecular cloning, which
are numerous and prone to complications and errors, the steps involved in genesynthesis are limited and straightforward. Synthesizing genes from design to execution takes between one to three steps, depending on the length of the sequence.

9Error-minimized: The fully automated nature of gene synthesis significantly reduces the possibility of unwanted errors.
Customizable: Whether used as a stand-alone technology or in combination withmolecular cloning, gene synthesis offers endless options for customization. Fromthe gene sequence and associated modifications to adaptability to diverse vectors,
the synthesis process is highly amenable to customization. Codon optimization is
one example of such capacity which can significantly increase protein expression inheterologous systems.

Enabling & Expanding Horizons of Synthetic Biology
The de novo synthesis of any nucleic acid sequence through gene synthesis enables
the creation of a variety of biomolecules. Partial or complete enzymes, genetic elements
and circuits, and metabolic pathways can be synthesized as core biomolecules forcellular assembly and incorporation. This significant feature of the gene synthesis
technology provides an enormous capacity for the development of foundational
compartments and tools for the field of synthetic biology.
As an emerging and interdisciplinary field, synthetic biology seeks to make thedesign, construction and optimization of biological systems easier, more predictable,
and reliable. The ultimate goal of synthetic biology is to enhance possible biological
functions for research applications and to develop therapeutics, materials, new means
for information processing and data storage, as well as new sources of food andenergy13, 14. This is achieved through the assembly of core biomolecules as engineeredparts towards new or improved biological functions, with the ultimate goal of helping to
improve living conditions and preserving the environment.
Knowledge and technologies from a wide range of disciplines, including engineering, biology, computer science, and mathematics have contributed to the developmentof this interdisciplinary field. However, among them, gene synthesis is considered themost enabling and makes up the foundation of synthetic biology. Information and tools
generated by this technology have made the transfer of engineering principles into the
biological discipline possible. Multi-part DNA constructs and biomolecular modules
used in DNA regulatory elements, gene and protein networks, obtained through the useof gene synthesis, are essential components of engineering-based circuits or network

10

designs in biological systems for modulating gene activity and cell behavior. Withextensive and direct applications in biomedicine, and implications or indirect impacts
on other scientific fields, gene synthesis technology is considered a revolutionary toolin biology research in specific, and in sciences in general.

GenScript’s comprehensive Molecular Biology Services, includes
gene synthesis, mutagenesis, cloning, and plasmid preparation; enabling
you to confidently move your synthetic biology ideas from design toapplication. Synthesize innovation today with GenScript!

11

Chapter Two
Applications

The revolutionary technology of gene synthesis has extensive and diverse applications
in basic research and applied sciences. Gene synthesis is a major foundation of thecurrent revolution in the biomedical sciences and biotechnology, impacting everythingfrom healthcare to conservation. Beyond life sciences, gene synthesis is enabling data
and material sciences to test theoretical hypotheses, conceive new technologies orapplications, and even develop new areas of science, such as DNA nanotechnology.
Ongoing improvements in synthesis fidelity and efficiency, process automation, cost
reduction and design algorithms will further expand the application of this technology.

Core Applications
The ability to design and synthesize custom nucleic acid sequences allows for threeunique core applications. These core applications can be used in several ways: directlyin an experimental setup/procedure, as the basis/precursor for the generation of othertools and reagents, or as a module in a multi-component tool or application.

1

Generating Nucleic Acid-Based Reagents

DNA- and RNA-based synthetic products of gene synthesis are used as essential tools
and reagents in biotechnology and biomedical research. These products range from
custom oligonucleotides, plasmids, genomic and eDNA, cDNAs and ORF clones as wellas interfering RNAs. The extent of the application of these reagents and tools in all areasof life sciences is so vast and fundamental that without them none of the current scientific advancements in disease pathogenesis, drug discovery and understanding life at
the molecular level would have been possible. Here are a couple of ways these tools areused in research today:
Oligonucleotides: As the most-widely used reagent, oligonucleotides are used ashybridization probes in bioassays, primers for nucleic acid amplification, geneassembly, sequencing, or for introducing mutations. Modifying these products withvarious tags, such as fluorophores, further expands their application in detection orquantification assays, such as quantitative real-time PCR. In recent years, advancements in high throughput synthesis technologies have further expanded the range
of applications for this valuable tool, offering hundreds of custom oligonucleotides
in library or array formats in single or pool versions for screening purposes.
cDNAs and ORFs: Synthesis of cDNAs and generation of ORF clones have helped
scientists overcome the shortcomings of molecular cloning and expand its utilization in science. Traditionally, an ORF clone is considered a plasmid that contains a
13

protein coding DNA insert which upon transfection into the right host, instructs themRNA translation machinery to express the full length, functional protein. Synthesisof an ORF skips the success-limiting step of obtaining the full-length gene sequence
through PCR and restriction enzymes in conventional cloning. Moreover, one caneasily assemble several gene cassettes to form fusion proteins while ensuring themaintenance of the correct open reading frame. Synthesized cDNAs and ORFs canbe delivered bare or cloned into any type of vector. While bare ORFs are less costly
and more suitable for quick high-throughput screenings as well as in vitro transcription and translation, they are less advantageous when compared to ORF clones.
Insertion of an ORF in a vector (a) protects the synthetic DNA from degradation, (b)facilitates clonal amplification in a transformation-competent host, and (c) simplifies
shuttling of the synthetic gene into a variety of vectors for transient of stable expression.
Synthetic short oligonucleotides and genes can be used as reagents for the synthesis of vectors, eDNA or the entire genome of an organism. For short eDNAs, a one-step
synthesis will generate a reagent for direct application in an experimental setup. For
large eDNAs or genomes, ORFs and regulatory elements are first synthesized and later,
with the use of enzymes or homologues recombination, assembled to create the wholegenome. Introducing these tools and reagents as functional units or modules of a
network into various biological hosts, allows the study of regulatory elements, transcription and translation machinery, function of DNA sequences outside the nucleus, andgeneration of new life forms.
RNA interfering molecules: RNA interference (RNAi) was originally named “posttranscriptional gene silencing” and “quelling” based on the initial discovery that
RNA inhibited protein expression in fungi and plants. The new terminology was latercoined in late 1990s where a similar phenomenon was discovered in C. elegans15.
Further work on elucidating the underlying mechanism of RNAi combined with thepower of gene synthesis, provided a plethora of reagents for studying mammaliangene expression and developing therapeutics. RNAi reagents, such as siRNAs,shRNAs, and miRNAs, are the preferred tools for loss of function studies, target
validation, and gene expression analysis in signaling pathways induced by natural
ligands or toxic compounds. RNAi molecules can be used directly or throughvectors in transfection assays in vitro and in vivo. Compared to dominant negative
constructs, ribozymes, or knock-out animal strategies, the RNAi approach is lessexpensive, more straightforward, efficient, and target-specific. Advancements indesign algorithms and modification technologies is leading to the development of
the second generation of RNAi tools with increased stability, greater specificity, andwith less chance of invoking an immune interferon response.

14

By synthesizing and cloning Rhinovirus RV1BV4 capsid protein and human
N-myrisotyl transferase, GenScript helped Bentley et al. demonstrate how theinteraction between these two proteins can induce an inflammatory immune
response in the respiratory system.
Recombinant Myristoylated Rhinovirus VP4 Protein Activates Pro-Inﬂammatory Gene
Expression in HEK293 Cells and Primary Alveolar Macrophages. Am J Respir Crit
Care Med, 2017.

2

Codon Optimization

Proteins are effector molecules in cellular machinery and as such, studying or modulating living organisms requires access to functional proteins in large quantities. Molecular cloning allows heterologous gene expression in which the protein coding sequence
from one organism can be transferred into another host or model organism for convenient expression and extraction of proteins in a large scale. Heterologous expression
enables the production of pharmaceuticals of human or animal origin without thehealth concerns associated with production from their native host. Unfortunately thisstrategy often suffers from lower than ideal rates of protein expression.
A common cause of low expression levels is codon bias or the variation in codon
usage frequency among different species. Translation of a heterologously-expressedgene can stall due to tRNA scarcity in the host organism which in turn leads to low
protein levels and increased rates of improper protein folding. While overexpressing
rare tRNAs can help remedy this problem to some extent, it can interfere with translational machinery that regulates all endogenous protein turnover and neglect to address
other optimizable features of the coding sequence. Taking advantage of codon degeneracy is the alternative and preferred approach: the DNA sequence can be changed ina way that does not alter the final amino acid sequence, while improving the efficiencyof transcription, mRNA stability, translation, and protein folding.
The process through which an amino acid sequence is rendered into a DNA
sequence with codon usage suitable for maximum expression in an organism is called“codon optimization”. Codon optimization not only helps with codon usage bias, butalso improves other features of a DNA sequence that affect the efficiency of transcription, proper splicing and processing of mRNA, and its stability. Optimizing codons canbe achieved through the laborious and costly method of side-directed mutagenesis,but application of gene synthesis can make this process efficient, fast, and less expensive16. Development of a variety of algorithms enables the design of the most optimized
sequence for increased expression, sometimes upwards of 100 folds when comparedto the non-optimized versions of the gene.
Codon optimization is widely used in different disciplines. For example, in structural
biology studies in basic science,15

codon optimization is employed to ensure sufficient yield of soluble proteins. In translational research and drug development, codon optimization helps with enhancing theimmunogenic response to DNA vaccines. Widespread application of codon optimization is also adopted in agriculture for a variety of purposes, such as improving crop traits
to increase yield or confer pest resistance.

Factors Influencing Protein Expression
2. mRNA processing and stability
DNA

Deviant folding

cryptic splice sites
mRNA secondary structure
stable free energy of mRNA

Natural folding

4. Protein foldingcodon context
codon-anticodon
interaction
translation pause sites
CGU

1. Transcription
cis-regulatory elements (TATA box,
termination signal, protein bindingsites, etc.)
chi sites

3. Translation
codon usage bias
ribosomal binding sites (e.g. IRES)
premature polyA sites

polymerase slippage sites

GenScript’s OptimumGene™ PSO-driven algorithm, patented in2012, efficiently optimizes sequences by simultaneously taking into
account 200 variables that are known to influence expression, resulting
in up to 100-fold higher protein yield than the native sequence and up
to 50-fold better results than other codon optimization design tools.
Improve your expression levels today!

16

GenScript’s Optimum™ gene optimization algorithm and gene synthesis
services, allowed Ramezani et al. to demonstrate that a sequence-optimized variantof the Pertuzumab gene can improve its production as a biosimilar drug.
Improving Pertuzumab production by gene optimization and proper signal peptide
selection. Protein Expression & Puriﬁcation, 2017.

3

Mutagenesis

Modification of DNA through mutagenesis is a well-established laboratory technique.
These mutations help with understanding the function of a protein or genetic pathway,or providing new proteins with novel or enhanced functions with potential for therapeutic and industrial applications.
Traditional methods for introducing mutations include subjecting live cells or
animals to UV radiation or chemical mutagens. These methods, though useful in reverse
genetic screenings, have several drawbacks: (a) they usually lead to high rates of lethality and low rates of useful mutant phenotypes, (b) they are random for nature and assuch require laborious efforts to investigate their outcome and make correlations to thelocation or frequency of the mutations, and (c) mutations may be biased and as a result
limit the range of possible mutants to only a fraction of those that are theoretically possible or that can be obtained through other strategies. An alternative approach is site-directed mutagenesis, which introduces variation at specific and desired locations withinDNA. This strategy, which is based on having prior knowledge of DNA sequence(s), canbe performed using PCR-based methods. Mutated primer sequences are first createdand used for introducing mutations through the amplification process, and finallyproducts are subjected to sequence verification. While this approach provides specificity and is less labor intensive, it still can be tedious, error-prone, and costly.
Generating mutations via gene synthesis overcomes the shortcomings of traditional
methods and provides new capabilities. Using this strategy, small or large number ofsystematic or random mutations can be created in various parts of a gene and regulatory elements, with higher efficiency and savings in effort and time. This results in moreexperimental control over the types or number of mutations in any mutant strain or
gene.
GenScript can deliver sequence-guaranteed mutants within two weeks. Save
your lab the hassle of conducting site-directed mutagenesis, sequence
verification, and error correction. Get your mutants on the timeline that best suits
your needs with GenScript’s Express Mutagenesis Services.

173.A. Gene Disruption Constructs
Targeted deletion of a gene, a series of genes, or regulatory elements of a gene is a
useful tool in understanding biological processes. Gene synthesis helps with the generation of custom gene disruption constructs that can be used for in vitro studies or to
generate mutant strains or knock-out animal models. Using this technology overcomesthe limitations of conventional methods, such as transposons, restriction enzyme digestion and PCR-based methods16. Specifically, the lack of adequate restriction sites,
non-specificity, and the requirement for large fragments of DNA can be a hindrance togenerating multiple or even single mutations. Assembly of smaller synthetic, mutant
gene compartments allows the construction of mutant gene cassettes for faster andmore efficient investigation of genomic sections. Synthesis of a variety of ORF clones
containing mutations or deletions that are expression-ready, facilitates the discovery of
new protein functions, generation of proteins with improved or novel functions, andinvestigating disease pathogenesis. Application of gene synthesis results in increased
accuracy when generating mutant constructs, as well as savings in both time andmoney during experimental execution.

3.B. Gene Variant Libraries
Gene synthesis facilitates the discovery or design of new protein variants (mutants) withproperties desired for medical, industrial, and agricultural applications. Gene variant
libraries allow for systematic, unbiased investigations, such as high-throughput screening for discovery biology, drug development, directed evolution for protein engineering, and in vitro molecular optimization to generate mutant proteins with improved ornovel properties14. A synthetic DNA library may also be used to construct novel genetic
circuits encoding enzymes that constitute biosynthetic pathways for metabolicengineering. They are also useful in identifying novel gene regulatory sequences or to
identify minimal domains critical for protein structure and function. Gene synthesis
allows the construction of a variety of custom libraries, such as site-directed mutagenesis, scanning point mutation, randomized and degenerated, truncation variant,precision mutant and combinatorial assembly libraries suitable for different experimental objectives. These libraries are fast becoming the preferred tool of choice for providing reliable, accurate, and reproducible results with time and cost efficiency.

18

GenScript’s expertise in de novo gene synthesis, and its proprietary

GenPlus™ High Throughput Gene Synthesis Technology, allow for thesynthesis of gene variant libraries of any size and complexity with
unprecedented efficiency. No matter your DNA library construction goal,
GenScript can provide a tailored service that meets your needs!

Applications of Core Products of Gene Synthesis
Type

Application

cDNA/ORF

Over-expression or heterologous expression forfunctional, enzymatic or structural studies

Customized coding sequences

Expressing fusion proteins
High-level protein expression from codon-optimized
sequences for functional, enzymatic or structural studies

Promoter-reporter constructs

Monitoring gene expression downstream of manipulations to transcription factors, signaling cascades, etc.

Genomic DNA

Creating synthetic genes or genomes to obtain novel
functions
Studying gene structure, regulation, and evolution

Mutant sequences

Functional studies (promoter-bashing, amino acidsubstitutions)Protein engineering (rational design or screening/directed evolution)

RNAi constructs

Gene regulation studies
Intercellular communication

Extracellular DNA

Biofilm formation
Intercellular signaling as in cancer metastasis

Gene variant libraries

19

Promoter optimization
Functional characterization of any gene or protein
Optimizing protein structure and function
Improving stability and affinity of enzymes
Optimizing antibody affinity
Creating and screening a large number of variants
when designing a metabolic pathway

Broad Applications
The core applications of the gene synthesis technology can be used for broader
applications in basic life sciences, medicine, engineering, and agriculture. These
applications not only provide novel solutions to existing and emerging problems facing
society and the environment, but also create and define new scientific areas.

1

Synthetic Biology

About thirty years ago, the discovery of restriction enzymes and their application inconstructing recombinant DNA molecules was predicted to lead us into the era ofsynthetic biology for constructing and evaluating new gene arrangements with unparalleled efficiency and accuracy17. Now, that prediction has turned into a vibrant andinterdisciplinary field that seeks to make the design, construction, and optimization of
biological systems easier, more efficient, reliable, and predictable. Using rational design
strategies, synthetic biologists modify, combine, and repurpose existing natural genetic
elements to create new parts and systems. Their approach is based on standardizationof genetic modules, decoupling of complex biological problems into simpler units thatcan be solved or pieced with other units to create entirely new systems; reducing
complexity and increasing efficiency of engineered biological systems18. The ultimate
goal is to use these novel synthetics towards deepening our understanding of natural
motifs and networks, developing new medicinal drugs and devices, new means of
information processing and data storage, as well as sources of food and energy that cansimultaneously help human health and preserve the environment.
Gene synthesis technology has enabled synthetic biology scientists to access highquality genetic material in large quantities, allowing for novel projects and applications
to be pursued. Using gene synthesis to create genetic building blocks and modules for
mixing and matching to build networks and circuits has fostered the development ofnovel methods and expanded the scope of gene synthesis applications. While early
synthetic biology research focused on engineering networks in prokaryotic hosts, it is
now the mammalian synthetic biology that is taking the most advantage of the powersof gene synthesis through manipulation of mammalian genomes and cloning large
DNA circuits. Biomolecular tools generated by synthetic biology include tools toregulate RNA, transcription, protein turnover and signaling pathways. These tools, suchas BiobricksTM, are offered as standard interchange parts that can be assembled in avariety of combinations to form complex systems and devices for diverse applications.

20

Broad Applications of Gene Synthesis ProductsCore Applications

Synthetic RNA/DNA

Mutagenesis

Codon Optimization

Synthetic Biology

Agriculture &Environment

Translational Research& Medicine

Food Production

Gene Therapy

Bioremediation

Diagnostics

Sustainable Energy
Production

Drug Delivery

Genomic &Genetic CodeEngineeringDNA
Nanotechnology& Smart Materials

Basic Science
Research21

Directed Molecular
Evolution

Regenerative
Medicine

Metabolic &Signaling Pathway
Engineering

Vaccines &Therapeutic
Antibodies

1.A. Metabolic & Signaling Pathway Engineering
The goal of metabolic engineering is to optimize microbial metabolic pathways toimprove product yield. However, with the application of synthetic biology the scope of
metabolic engineering has greatly expanded to not only increase yield in a more
efficient and cost-effective manner, but also to produce a wider range of metabolites
with unique features. Following the main components of biology’s central dogma –transcription, translation and RNA processing – molecular control over metabolic andsignaling pathways is first studied and quantified. This is then followed by the designand synthesis of either improved natural parts or completely novel elements, which are
inspired by various natural components from various organisms or pathways. Assemblyof these synthetic parts into circuits and networks can ultimately lead to engineered
systems that can be incorporated into a new host to provide the desired metabolic
output.

In addition to quantification studies on the rate-limiting steps and substrate concentration, synthetic biology approaches have also been focused on engineering the following aspects of protein expression for adjusting cellular metabolism19:
Promoters for regular or constitutive activation through mutagenesis, recombination, or assembly of components taken from different organisms and pathways.
Given that mutations often interact in a cooperative and non-linear manner withregard to metabolite production20, synthetic biology has enabled multiple and simultaneous mutations in several genes involved in the transcriptional machinery in an
approach currently called the Global Transcription Machinery Engineering (gTME).
Ribosome binding sites (RBS) based on thermodynamic models of translation
initiation which have led to the development of an RBS Calculator. Based on thedesired translation initiation rate, gene sequence, and host organism, this algorithm
predicts a customized RBS to achieve a targeted translation initiation rate21.
RNA-focused parts to modify RNA degradation affecting steady-state expression
level. These parts include modular RNA elements, RNA regulators, and RNA aptamer
domains. Moreover, RNA-related sequences can also be engineered. A prominent
example is the Tunable Intergenic Regions (TIGR), which are synthetic RNA
constructs that include two hairpins joined by an RNase cleavage site, and can beused to connect co-transcribed ORFs22.
Spatial organization of cellular components of a pathway to increase the local
concentration of pathway intermediates and exclude competing cytosolic
pathways. RNA and DNA scaffolds are examples of such engineering attempts23, 24, 25.

22

Combinatorial application of these tools allows the generation of chimeric and novel
pathways for optimized interaction in a heterologous host to achieve metabolic goals.
In addition, synthetic biology approaches are used to engineer membrane receptors
and protein domains involved in enzymatic activity, protein-protein interactions todevelop networks for detecting unnatural small molecules, stimulating the immune
system, and enhancing metabolic control14. The executive host of such approaches,
which are kept in their natural state or engineered, can be isolated mammalian or plant
cells as well as prokaryotes in single or ecosystem form. Engineering microbial ecosystems is currently being tested for a variety of purposes, such as bioremediation andtherapeutic purposes26.

Engineering Biology Through Synthetic Biology
Systems

Devices

Parts

DNA/RNAGTACGAATGGCAGTCCTTGGCAAObjective

Transcription
al Control

ProteinTurn-Over
Regulation

RNA Control

Genome &PathwayEngineering

GenScript is proud to be a contributing partner in the Sc2.0 International
Consortium whose goal is to build a designer synthetic eukaryotic genome of
Saccharomyces cerevisiae.
Design of a synthetic yeast genome. Science, 2017.

23

1.B. Genomic & Genetic Code Engineering
The precise manipulation of one or more genetic loci or amino acid codons for a specific outcome is the main objective of gene and genomic engineering. Defining thedesired outcome is application specific and includes correcting deficiencies, imparting
novel functions, or de novo synthesis of a new system or organism. Synthetic biology
has helped to improve the specificity of such engineering objectives. Moreover, it has
opened up new avenues in design and application, which would have been impossible
to conceive and implement in the past.
Precise manipulation of mammalian genomes was first made possible by site-specific recombination. This method, based on bacterial or fungal recombinases (such as Cre
recombinase) catalyzing the recombination of a pair of short target sequences (such as
LoxP sites), can lack specificity, though. Using synthetic biology, conditional geneknockouts with increased specificity can be created. For example, an expression
cassette containing two fragments of a split Cre recombinase from two different
tissue-specific promoters has been synthesized. This allows the induction of aknock-out response to a combination of two sequences representing the same gene toachieve higher specificity27, 28. Moreover, synthesis of programmable DNA nucleases,
such as TALENs and CRISPR, that can introduce site-specific DNA breaks leading tolocalized homologous recombination, has greatly improved the efficiency of genomic
engineering. Furthermore, these methods offer the possibility of simultaneous allele
mutations, although with this comes an increase chance of off-target cleavage. Recent
advancements in Multiplexed Genome Engineering (MAGE) has been shown to reduce
this chance. This oligonucleotide-mediated recombination approach allows for editing
multiple genetic loci in parallel and is demonstrated to be useful in improving the yieldof industrial product29. Similar attempts are under way in mammalian and plant systems
for potential gene therapy and genetic improvements in crops, respectively30. Application of MAGE in microbial populations, which enables the generation of divergent andcomplementary phenotypes to obtain engineered ecosystems with desired functions,is also underway26.
Engineering the genetic code is another emerging area whose conception wassolely based on the gene synthesis and synthetic biology techniques. This area iscomprised of (a) codon swapping through changing the genome sequence, such assynonymous codon swaps in one or more genes, (b) codon introduction throughassigning a new amino acid to a codon without removing its original function, such as
UAG usage as a stop codon while maintaining its coding for selenocysteine and pyrrolysine, (c) codon reassignment through changing the amino acid assignments of one ormore codons across the whole genome, and (d) codon creation through adding a newcodon to the translation code table, such as using codons with unnatural bases orquadruplet codons31. Engineered genetic codes can help with overcoming thechallenges of the universal genetic code (such as viral infection and undesired horizontal gene transfer), understanding origins of life, and providing potential for new anduseful protein functions with diverse applications.
24

Advances in gene synthesis and synthetic biology in fabricating long DNA sequences have also paved the way for current efforts towards de novo synthesis of wholegenomes. With the experience gained through the successful construction of mousemitochondrial genome32 and a bacterial minimal genome33, a consortium of scientists
are currently working on synthesizing the modified version of yeast genome34. Suchcustom-made organelles and organisms are used to further understand the fundamentals of life and develop novel tools and products.

The International Genetically Engineered Machine (iGEM) competition is thepremiere Synthetic Biology competition, with separate divisions for high school
students, undergraduates, and researchers/entrepreneurs. Teams use
standardized biological parts, both existing and new, to build novel biologicalsystems and operate them in living cells, tackling real world problems facing
society today. GenScript is proud to sponsor iGEM teams and to provide genesynthesis services that allow competitors to bring their designs to life!

1.C. Directed Molecular Evolution
Another area where synthetic biology has helped advance is artificial evolution. Directed molecular evolution aims to use a variety of synthetic biology-made parts andcircuits to elucidate gene and protein function, improve protein and pathway activities,
and fabricate novel proteins. From simple mutagenesis to shuffling receptor domains
and manipulating ligand binding specificity, artificial evolution is providing a short-cut
for obtaining enzymes, receptors, pathways and products. Development ofhigh-throughput techniques, such as MAGE, are further advancing the screening ofartificially-evolved molecules for therapeutic and industrial applications14, 16.

1.D. DNA Nanotechnology & Smart Materials
One of the main problems in our times, currently being dubbed the information age, isthe storing of data generated worldwide, which is predicted to reach 40 zigabytes (ZB)
by the year 202035. With a perspective of using smaller mediums rather than bigger data
storage centers, the key to solving this issue can be found through the use of the DNA
molecule. Billions of years of evolution have optimized DNA for transmission andstorage of data in a way that is both efficient and reliable. Theoretically, all 40 ZB of
man-made data can be stored in just 90 grams of DNA36. Initial attempts to use DNA as
an archival medium could go only so far because of the difficulties faced when attempting to synthesize long stretches of DNA. However, with the current technologicaladvancements in DNA synthesis and sequencing, synthetic biologists are fast making
progress in storing large datasets in DNA. Although current limitations such as lack ofrandom access read, rewritability features, and ability to decode data in the distant25

future is keeping this novel solution at the experimental level, the multitude of advantages the DNA-based storage offer is foreseen to reshape the future of communicationand data storage in the near future36, 37.
Similarly, advances in synthetic biology has allowed the realization of an array oftheoretical ideas in the field of DNA nanotechnology. Pioneering work of Seeman in theearly 80’s, which established the tile-based assembly method38, combined with theorigami method put forth by Rothemund at the turn of this century39 have facilitated thefabrication of a variety of DNA nanostructures, such as nanosensors, nanoelectronic,
nanophotonic devices and scaffolds. These programmable materials can be built in twoor three dimensions and serve as a useful tool to construct structures with a variety ofsizes and complexity with applications in biosecurity, product barcoding, protein
structure studies, basic science research, diagnostics, and therapeutics40.

Advantages of DNA-Based Data Storage
c

G

D

F

F

D

T A
F

Reproducible through fast and cost-effective PCR

D
F

c

G

D

F
D

Stable and not subject to sequence change

F

Passive and cost-efficient maintenance

T A
F

c

G
D

Hack-proof based on lack of remote access

D
F

D
F

G

c

T A

Storage capacity 1,000,000-fold higher than current
platforms

Long lifespan across thousands of years

DF

Devoid of technological obsolescence

1.E. Improving Traditional Research Methods
While basic science research was the birth place of synthetic biology, it has been thefirst field to benefit from its existence. One of the earliest applications of synthetic
biology in basic science research was the use of transcription factor circuits in studying
gene expression and regulation. The first generation of these circuits was comprised of
a combination of natural and synthetic parts. For example, the LacI circuit, which is stillwidely used in laboratories, consists of a naturally existing bacterial transcription factor
lac repressor (LacI) and a synthetic promoter containing the LacI transcription
factor-binding sequence. This synthetic circuit can sense the expression of specificendogenous transcription factor as an input signal upon binding of the small molecule
isopropyl-β-d-thiogalactopyranoside (IPTG). Alternatively, the promoter sequence of an
endogenous gene can be fused upstream of a synthetic transcription factor coding
sequence to drive its expression.

26

The second generation of transcription factor-based circuits, comprised of synthetic
programmable transcription factors, is fast replacing the early types as a result of thehigh level of customization they offer. These include Zinc Finger-containing Transcription Factors (ZF-TF), Transcription Activator-Like Effectors (TALEs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based regulators. Customizationof various elements of these systems has greatly expanded the repertoire and specificityof their application in basic science studies. Moreover, by fusing enzymes such as DNA
methyltransferases that catalyze the addition or removal of chromatin marks, further
studies on histone modification and ultimately gene regulation is made possible41, 42, 43.
Synthetic gene circuits comprised of RNA and transcription factor elements areanother synthetic biology-generated tool with basic science application. These circuits
help with testing reverse engineering algorithms with positive and negative feedback
loops to reveal the highly interconnected network of gene regulatory elements and tocontrol cellular behavior44, 45. Moreover, rebuilding synthetic signaling networks andstudying them in an exogenous context, such as expression in a heterologous host,
simplifies the study of the complex and interconnected endogenous signaling
pathways that may compete or affect a given network14.
In addition, development of novel high-throughput methods through the synthesisof tandem repeats of short DNA sequences in libraries have expedited the discovery ofstrong transcription factor-binding motifs or gene and protein regulatory networks. For
example, barcoding promoter variants with short DNA sequences and measuring theiractivity by high-throughput RNA sequencing, named ”massively parallel reporter
assay”, has allowed the study of a large number of variants of enhancers and helpedwith the discovery of conserved regulatory motifs within enhancers46.

Codon optimization, gene synthesis and cloning services of GenScript helped
Zhang et al. to engineer the microalgae Synechocystis for generating an
algal-based biofuel.
Construction and application of the Synechocystis sp. PCC6803-ftnA in microbial
contamination control in a coupled cultivation and wastewater treatment. J of
Environmental Sciences, 2016.

27

2

Translational Research and Medicine

A common goal among scientists in different disciplines is to develop novel approaches
for disease diagnosis and treatment. Achieving this goal has been made more practicalthrough the application of gene synthesis and its off-shoot, synthetic biology. Throughthe expansion of biological systems regulation and improving safety and efficiency ofengineered systems, we can now develop novel and more efficient vaccines andantibodies, drug delivery methods, tissue and organ repair strategies as well as cell andgene therapy approaches.

2.A. Diagnostics
Access to easy-to-use, reliable, and time-saving methods for disease diagnosis has
always been the ultimate goal of diagnostics and its associated fields. The arsenal oftools generated by gene synthesis is beginning to facilitate and expedite such attempts,
and also bring many futuristic ideas into reality. Diagnostic tests take advantage ofengineered RNA and DNA as well as their associated synthetic circuits. For example,
engineered gene expression systems can now be coupled with synthetic RNA switches
and linked to a colorimetric output detection, the combination of which results in avisual output only in the presence of a specific biomarker or pathogen. Such systems,
printed on paper for portability, are currently being tested for detection of antibody
resistance and viral infection47, 48.

2.B. Gene Therapy
Gene therapy involves the delivery of therapeutic genes packaged within viruses into
the diseased tissue of interest. Gene synthesis is helping advance gene therapy on
several fronts: engineering efficient therapeutic genes, improving the specificity ofgene delivery, and efficient expression of therapeutic genes in the patient. Cell or tissue
specificity is achieved by expressing therapeutic genes from actively expressed cell or
tissue-specific promoter elements whereas the combination of virus variant libraries
and in vivo-directed evolution can identify the most efficient virus for gene delivery49, 50.
This approach can also be combined with autologous cell transplantation for enhanced
efficacy. The utilization of synthetic biology and its tools is allowing for the removal ofstem cells from a patient, correction of defected genes with the use of programmabletranscription factors, and then transfer of corrected genes back to the same patient withthe help of engineered viruses. The CRISPR-Cas9 genome editing, in particular, is quickly growing into the main and most efficient choice for a variety of gene therapies fortreating genetic diseases and cancer51, 52. The advantage of CRISPR is in its versatility,
flexibility, and specificity: versatility in the use of an RNA molecule to specify a target
rather than a protein domain, flexibility in designing libraries of transcription factors, andspecificity in targeting the right sequence.

28

2.C. Cell Therapy
Cell therapy, which is now at the forefront of modern therapeutic interventions, is also
benefiting from gene synthesis and synthetic biology. In particular, application ofsynthetic biology in Chimeric Antigen Receptor T-Cell (CAR-T) therapy is demonstrated
to be successful in treating tumors that were not responsive to other forms of therapy. In
this strategy, synthetic CAR libraries help identify the best epitope, T-cells areengineered to express the identified epitope ex vivo, and then engineered cells aretransferred back into the patient to destroy the diseased target cells53. While the majority of existing CAR-based therapies target cancer cells based on the presence of a single
cancer-specific antigen on any cell, emerging approaches are focused on improving
specificity through the application of “AND logic gates”. By requiring the presence of
two antigens or one antigen and a small molecule drug, such systems ensure the targeting of specific cells only14.

2.D. Vaccine & Therapeutic Antibody Development
Synthetic biology approaches that involve bioinformatics, gene synthesis, and mammalian or plant cell line production systems are giving rise to the production of the second
generation of vaccines and therapeutic antibodies. Discovery and engineering of therapeutic antibodies is greatly advanced through the use of high-throughput screenings
as well as DNA and mRNA display technologies powered by synthetic biology. Similarly,
development of improved conventional as well as novel DNA- or RNA-based vaccines
has been made possible through the application of synthetic biology tools54, 55. In silico
design of novel immunogens, comprehensive viral sequence libraries, and RNA andcodon usage modifications for improving antigen expression enable the developmentof vaccines with improved safety profiles, increased immunogenicity, and an enhanced
breadth of immune responses56, 57. In particular, combinatorial application of several
gene synthesis and synthetic biology approaches in the development of RNA-based
vaccines offers promising therapeutics. These vaccines, which are used for conferring
immunity against pathogens and cancers lack the ability to mutate the genome58.
Therefore, they are preferred over DNA vaccines for safety concerns. The design ofRNA-based vaccines through engineered immune cells is also foreseen to help
overcome systemic immunosuppression and increase efficiency59.

2.E. Regenerative Medicine
The emergence of the field of synthetic biology has coincided closely with the rise ofregenerative medicine, also called tissue engineering. However, until recently theenormous potential of applying synthetic biology in tissue engineering was
overlooked. Regenerative medicine’s main goal - to regenerate damaged tissues
through developing biological substitutes - has benefitted greatly from synthetic
biology’s tools60. This has been achieved through the biosynthesis and controlled
release of therapeutic molecules, synthesis of scaffold materials, regulation of stem

29

cells, and programming cells that can organize themselves into novel tissues.
DNA-based hydrogels as scaffold for tissue repair, synthetic multi-part signaling
networks as patterning elements61, and three-dimensional printing of living tissues
capable of displaying cellular behavior and physiology62 are a few examples of theapplication of synthetic biology in tissue engineering.

2.F. Drug Delivery
Tools generated by the gene synthesis technology are also being incorporated into thedesign of smart drug delivery systems to increase the drug targeting efficacy while
reducing side effects and toxicities of payloads63. These systems, which take advantage
of prokaryotic and eukaryotic cells, achieve a comparable therapeutic effect with much
lower dosage of a drug, offer a less invasive route of administration with gastric
enzyme-sensitive drugs, and provide a more cost-effective treatment through the in vivo
production and delivery methods64. Another application is in developing prosthetic
networks, which are synthetic circuits capable of sensing disease-relevant metabolites
for coordinating diagnostic, preventive, and therapeutic responses65, 66. These networks
are implanted in biocompatible genetically engineered encapsulated cells anddelivered into the body. Upon sensing imbalanced levels of a metabolite or marker,
such as high glucose levels, these cells can send a diagnostic signal, such as light, andrelease the right dose of a therapeutic drug, such as insulin.

Process of Engineering & Validating Novel Therapeutic Proteins
Select protein of interest; Definefeatures to optimize

In silico computational modeling ofmutant proteins to select lead candidates

High-throughput synthesis ofcodon-optimized mutant genes

Protein purification forbiochemical assays

Cell-based
functional assays

Pre-clinical/clinical drug
development

30

Expression of the E6 HPV oncoprotein, synthesized and cloned by GenScript, in
293 cells enabled Xu et al. to compile a comprehensive transcriptional andsplicing signature of its expression towards better understanding of the cervicalcancer pathogenesis.
A transcriptomic landscape of human papillomavirus 16 E6-regulated gene expression
and splicing events. FEBS Lett, 2016.

3

Agriculture and Environment

Application of gene synthesis in plant sciences has far exceeded its original use inunderstanding plant cell biology, physiology and signaling pathways, or discovering
plant-derived pharmaceuticals. Now, gene synthesis is enabling scientists to develop
new and sustainable forms of energy, improve the quality and quantity of plant andanimal-based sources of food, and remediate the detrimental effects of environmental
pollutants.

3.A. Food & Sustainable Energy Production
Successful gene manipulation and transfer of recombinant or exogenous genes tolaboratory animals in the early 1980s promised broader applications in plant andanimal agriculture67. Decades later and with the help of gene synthesis, those promises
are fulfilled not only at an exponential pace, but are also expanded into novel andcreative directions. In plant agriculture, manipulation of existing genes and introduction of synthetic transgenes of plant, viral, or bacterial origin into a variety of crops havesignificantly improved crop yield through modulating photosynthesis and resistance tomultitude of biological or environmental stress factors. Also, modulation of signaling
pathways through gene synthesis has conferred novel nutritional values, and improved
the natural health value and flavor of many plant-based sources of food68, 69. Transgenic
plants are also extensively used for the production of antibiotics, growth hormones,
enzymes, vaccines, and other therapeutic or diagnostic molecules70. Moreover, manipulation of pathways involved in the synthesis of starch, cellulose, hemicellulose, andoils has led to the generation of biofuels with minimum environmental footprint71.
Similarly, application of gene synthesis in animal sciences allows for altering the genetics and physiology of livestock species to increase the yield and dietetic value of
produced milk and meat72.

GenScript codon-optimized and synthesized the scorpion toxin gene LqhIT2 tohelp Tianpei et al. confer pest resistance to rice.
Scorpion peptide LqhIT2 activates phenylpropanoid pathways via jasmonate toincrease rice resistance to rice leafrollers. Plant Sci, 2014.

31

2.B. Bioremediation & Environmental Preservation
Conventional approaches for solving environmental pollution, caused by accidental
spills or poor human management, are found to be ineffective and costly. Application ofgene synthesis and its products, however, have facilitated the development of novel
techniques that are more effective, faster, less expensive, and more environment-friendly. One method is bioremediation that is based on degradation or immobilization oforganic and non-organic pollutants by engineered microorganisms. Using synthetic
vectors, DNA and RNA libraries, and optimized or novel gene sequences, regulatory
systems are built and engineered into microorganisms such that they could both sense
and process contaminants. Functionality of these systems is based on the production
and display of extracellular enzymes or proteins on the outer cell membrane that canmonitor and catalyze or trap a specific category of contaminants73. A similar applicationof gene synthesis is also used to engineer organisms capable of producing chemicalcompounds that are biodegradable and less taxing on the environment in comparison
to conventional sources of chemicals74. A prominent example is an engineered strain of
E. coli with the ability to synthesize 1,4-butanediol as a precursor for the manufacturingof plastics, rubber, and solvents75. The production of this non-natural chemical, basedon the construction of a novel pathway with no “blueprint” in nature using gene synthesis, helps preserve the environment through decreased dependency on oil-based
chemicals. Development of engineered crops for the generation of biofuels or withmodified physiological features with less dependency on water, carbon or nutrientsalong with bioengineering ecosystems are among the other benefits of applying genesynthesis in preserving the environment74, 75.

32

Chapter Three
Technology

The chemical synthesis of genes and genomes involves four major steps. This process
starts with identifying the application of the synthetic gene followed by the design andsynthesis of short oligonucleotide sequences for further assembly to compile the wholegene. The third step is quality control to verify the sequence and the last step is tocorrect possible errors before the synthesized sequence is prepared for its downstream
application. While advancements in chemistry and methodology have improved theefficiency of each step since the early years of the gene synthesis technology over 30
years ago, yet the core of each step has remained the same. This chapter provides abrief overview of the process behind the current, commercial gene synthesis technology.

The Process of Gene Synthesis

DNA Sequence
Selection

Sequence Optimization
and Oligo Design

Transformation,
Transfection, Downstream
Application

1

Oligo Synthesis

Gene Assembly

Quality Control:
Sequence Verificationand Error Correction

Cloning into
Vector of Choice

Sequence Design & Optimization

The first step in synthesizing a gene of interest is designing the right sequence. In thedesign of a synthetic sequence two aspects need to be taken into consideration: (1)downstream application of the synthetic gene, and (2) the synthesis and assembly
steps.

1.A. Downstream Application Optimization
Keeping the ultimate application of the synthetic gene in mind is an essential part of thedesign since placement of each nucleotide within the sequence can directly affect thesuccess of the downstream application. The following is a list of examples that
illustrates the importance of sequence design and optimization:34

In applications involving maximum heterologous protein expression levels,
sequence design has to consider codon optimization. In the contrary, if the synthetic
gene is to be used for studying endogenous gene regulation, codon manipulation
should be avoided.
When the synthetic sequence contains multiple segments, sequence design needs
to ensure that throughout the entire coding region the reading frame is maintained.
In cases where short flanking sequences are added to the gene sequence in order tofacilitate later excision or recombination, careful attention needs to be given to avoid
including or introducing restriction enzyme recognition sites or other sequencesthat might interfere with downstream workflow.
Presence of biologically functional domains, such as cis-regulatory elements or
RNase splice sites, may hinder in vivo assembly, maintenance, or expression of thesynthetic gene. Therefore, sequences coding for such domains have to be avoided
during the design step.

1.B. Synthesis Optimization
After finalizing the sequence design with regard to downstream applications, thesequence is analyzed to ensure a successful assembly. Given that synthetic genes are
made up of assembled short oligonucleotide sequences of 40-200 bp, sequence analysis helps determine the best way to divide the whole gene into fragments that are easily
synthesized and then seamlessly assembled (see step 3 below). In addition, somesequence features make the process of gene synthesis more challenging. Thesefeatures include extremely high or low GC content, highly repetitive sequences,complex secondary structures, unstable structural elements, polyA stretches, and very
long sequences over 1 kb.
Considering all the above-mentioned factors in the design of oligonucleotides can,therefore, be a confusing, time-consuming, and difficult process. Computational
software for designing and optimizing oligonucleotides can significantly facilitate thisprocess. Sophisticated algorithms incorporated in a variety of oligonucleotide design
tools take all factors affecting downstream application, synthesis and assembly steps
into consideration to ensure the synthesis of a functional sequence with minimum cost
and turnaround time.

35

Oligonucleotide Design Tools

Design Tool

Advantages

Limitations

DNA Works
http://helixweb.nih.gov/
dnaworks/

Easy to use. Predicts thepotential for oligo
mishybridization andsecondary structures.

Limited to PCR-based
methods. Scores based on
simulations may notcorrelate actual assembly
success.

Gene2Oligo
http://berry.engin.um
ich.edu/gene2oligo/

Simple user interface,designs oligos for both
LCR and PCR-based
assembly.

Limited to genes less than
1 kb in length.

GeneDesign
http://baderlab.bme.jh
u.edu/gd/

Breaks multi-kb
sequences into ~500
bp blocks for initialassembly in separate
pools.

Does not offer
mishybridization analysis.

TMPrimehttp://prime.ibn.
a-star.edu.sg

Designs oligos for both
LCR- and PCR-based
assembly. Produces themost homologous
melting temperatures (∆Tm < 3°C) and widest
range of annealing
temperatures (50–70°C).

Requires thoroughunderstanding of many
input parameters forcorrect submission; lessuser-friendly interface.

By considering a variety of critical factors involved in the different stagesof protein expression, such as codon adaptability, mRNA structure, andvarious cis-elements in transcription and translation, GenScript’s
proprietary OptimumGene™ provides you with the ideal sequence tomaximize protein expression in any host. Take the guess work out of
optimization with GenScript’s industry-leading gene optimization
service.

36

2Synthesis

In principle, DNA fabrication is oligonucleotide synthesis through a cyclical process inwhich nucleotide monomers are linked together in step-wise, chemical reactions.
Synthesis is traditionally carried out in a solid phase in the form of a column, although
the recently developed microfluidics and microarray platforms are increasingly becoming commonplace. The solid support, which is usually glass or polystyrene beads,
serves as an anchor for the growing oligonucleotide chain. The first monomer is
attached to the solid support through its 3’ OH (hydroxyl) group, then incoming monomers, based on the designed sequence, are added sequentially until the desired lengthis obtained and the chain is cleaved off of the solid phase.
The most commonly-used synthesis technique is the “phosphoramidite synthesis
method”, which was developed in the 1980s77and is now fully automated and robust.
Whereas DNA synthesis in living cells always occurs in the 5’ to 3’ direction, thephosphoramidite synthesis proceeds in the 3’ to 5’ direction. Moreover, the building
block nucleotide monomers used in this method are modified versions of the naturally
occurring nucleotides, and are called “nucleoside phosphoramidites”. Since natural
nucleotides and their phosphodiester analogs are not reactive enough for a fast andhigh-yield rate of inter-nucleosidic linkage formation, using 3'-O-(N,N-diisopropyl
phosphoramidite) derivatives improves the efficiency of synthesis. The functionalgroups of phosphoramidite monomers as well as any intermediate products generated
throughout this cyclical method, are conjugated with a variety of protecting groups to
avoid undesired side reactions. For example, the 5'-hydroxyl group of the first and allincoming monomers is protected by an acid-labile 4,4'-dimethoxytrityl (DMT) group.

The phosphoramidite synthesis method involves four main steps, which are cyclicallyrepeated until the desired oligonucleotide chain is synthesized. These steps are:
1. Deprotection: The protecting DMT group on the first monomer attached to thesolid phase is removed by a mild acid wash to expose the 5’ hydroxyl group for the
coupling reaction;
2. Coupling: The second monomer with its DMT-protected 5’ hydroxyl group and 3’hydroxyl activated by the conjugated phosphoramidite group enters the reaction.
Coupling occurs when the 3’ hydroxyl group of the second monomer forms a
phosphate triester bond with the 5’ hydroxyl group of the first nucleotide;
3. Capping: Uncoupled 5’ hydroxyl groups of any unreacted nucleotides areblocked by acylation to prevent later growth of an incorrect sequence. Typically,
acetic anhydride and dimethylaminopiridine are used which will have no effect onterminal nucleotides that are protected by DMT;

37

4. Oxidation: By adding iodine, the relatively unstable phosphite triester inter-nucleotide bonds are converted into the phosphodiester bond that forms the familiar
backbone of DNA.
Once the synthesis of the desired chain is complete, the protecting groups are removed
by treatment with a strong base such as ammonium hydroxide, and the 5’ end of theoligonucleotide is phosphorylated. Prematurely terminated strands are also removedby purifying the eluted product via gel electrophoresis and cutting out the band withthe correct length. Synthetic oligonucleotides are usually further characterized bysequencing or are simply subjected to mass spectrometry to verify the predicted molecular mass.

Phosphoramidite Oligonucleotide Synthesis Reaction Cycle
Terminal,
Protected Monomer

Repeat Steps 1-4
until chain iscomplete

Step 4:

Step 1:

Deprotection

Phosphoramidite
Reaction Cycle

Terminal,
De-protected
Monomer+

Activated
Monomer

Oxidation

Step 2:
Coupling

Step 3:
Capping

Unreacted
terminal
Monomers
are acetylated

Solid phase phosphoramidite chemistry can generate oligonucleotides with lengths <100 nucleotides with coupling efficiencies of almost 99%78. The automated process
allows the synthesis of 96-384 oligonucleotides simultaneously at 10-100 nmol
scales79. Over the last few decades, novel modifications in each step of the synthesis
process as well as improvements in raw materials, have led to the development of
improved methods to decrease the amount of starting materials, reagents, and waste
needed to produce oligonucleotides, resulting in a more affordable and productive
38

process with both faster speeds and greater yields. Emerging microfluidics and microarray platforms used in high-throughput gene synthesis are among such advancements80. Microarray technologies are based on spatially confining reagents and directing reactions with very fine spatial and temporal control. Such methods rely on using
inkjet printers to dispense picoliter reagents to specific locations on a silica chip,
controlling the deprotection step with light-activated photochemistry in a microfluidic
system. These technologies also use programmable microelectrode arrays to direct
redox reactions at desired spots. In practice, however, these methods can create error
through imprecision and can introduce new costs, such as the expense of manufacturing unique photomasks required for photolithography platforms.

High-Throughput Array-Based Gene Synthesis Technology

GenPlus™ High Throughput Gene Synthesis Technology is GenScript's latest
breakthrough: a proprietary technology platform combines parallel synthesis
and automation to enable an unprecedented capacity of 100 Mbp per month;
the equivalent of up to 100,000 genes, or 20 bacterial genomes! Tackle genesynthesis projects of any size with GenScript’s industry-leading gene synthesis
expertise today!

39

3

Assembly

The chemical synthesis of oligonucleotides can efficiently generate sequences of up to120-150 nucleotides. However, since the synthesis of oligomers with longer lengths areassociated with lower yield and higher error rates, it is recommended to fabricate longsequences through assembling shorter sequences. In the past several decades, anumber of assembly methods have been developed. These techniques are ligation-mediated, PCR-mediated, or a combination of both methods. Increasingly, recombination-mediated methods are also used to simultaneously assemble and clone long DNA
sequences for increased efficiency and reduced error generation.

Main Assembly Methods for Synthetic Genes & Genomes

Oligo Assembly Methods

Reference

POLYMERASE-BASED
Dual-Asymmetric (DA) PCR

Sandhu et al. (1992)88

Overlap Extension (OE)
Polymerase Cycling Assembly

Horton et al. (1993)89

Asymmetric PCR

Wooddell & Burgess (1996)91

Thermodynamically-Balanced Inside-Out (TBIO)

Gao et al. (2003)81

Two-Step (DA+OE)
Microchip-Based Multiplex Gene Synthesis

Young & Dong (2004)92

One-Step Simpliﬁed Gene Synthesis

Wu et al. (2006)94

Parallel Microﬂuidics-Based Synthesis

Kong et al. (2007)95

Single Molecule PCR

Yehezkel et al. (2008)96

TopDown Real-Time Gene Synthesis

Huang et al. (2012)97

Stemmer et al. (1995)90

Tian et al. (2004)93

LIGASE-BASED
Shotgun Ligation

Eren & Swenson (1989)98

Two-Step Ligation and PCR

Mehta et al. (1997)99

Ligase Chain Reaction

Au et al. (1998)100

Brick-Based

Kelly et al. (2009)101

RECOMBINATION-BASED
Sequence-and Ligation-Independent Cloning (SLIC)

Li and Elledge (2007)102

Transformation-Associated Recombination

Gibson et al. (2008)103

BioBrick Assembly in E. coli

Ho-shing et al. (2012)104

40

The following is a list of major categories of early and current assembly methods with afew example techniques in each group.

3.A. Polymerase-Mediated AssemblyThis category of assembly techniques are the most commonly used, and rely on thepolymerase chain reaction to extend and assemble overlapping oligonucleotides into
double-stranded fragments by cycling in a non-exponential process79. Assembly inthese techniques can be single-step in which the desired gene product is assembled ineither a single reaction or in multiple enzymatic reactions. Careful application ofoligonucleotide design software packages is essential in the success of the assembly
step since each method is based on unique primer sets. Polymerase Cycling (or Chain)
Assembly (PCA)-based method is one standard technique in this category. In thistemplateless PCR, all of the single-stranded oligonucleotides are combined into a single
tube and are subjected to thermocycling with the use of the outermost primers to amplify the full-length sequence. The success of this method depends upon the accurate
synthesis of oligonucleotides designed to possess sufficient regions of overlap, similar
melting and annealing temperatures, and minimal opportunities for mis-hybridization.
In another multi-step method, called Thermodynamically Balanced, Inside-Out (TBIO)
assembly, overlaps between primers are balanced in order to obtain uniform annealing
temperatures across the assembly. Besides this novel approach to primer design,
primers are added in a concentration gradient in which the innermost pair has thelowest concentration and the outermost pair the opposite. The selective pressure for
amplification will lead to the assembly of longer products, favoring the fabrication of the
full-length, desired gene81.

3.B. Ligation-Mediated Assembly
Ligation-based assembly techniques take advantage of the enzymatic activity of bacterial ligase for joining complementary overlapping strands to produce larger DNA
fragments. This category represents the earliest methods used in gene synthesis,
including the genes for yeast alanine tRNA and human insulin A synthesized in the late
60s and 70s, respectively82. These methods were initially carried out sequentially, butaccess to higher quality oligonucleotides, application of thermostable ligases such as
Pfu DNA ligase, and development of methods for producing and selecting for circular
DNA have enabled a one-pot assembly of gene fragments79. An example method in thiscategory is the Ligase Chain Reaction (LCR) assembly, which involves a temperature
cycling reaction similar to PCR. In this technique, oligonucleotides are synthesized insuch a way that their 5’ end is phosphorylated and each contain overlapping sequences that span both strands of a desired DNA duplex. Once these oligonucleotides are
mixed together, they are heated to denature, and then cooled to promote both theannealing of oligonucleotides as well as their ligation by the Pfu ligase. The newly
assembled intermediate products can then serve as templates for additional ligation

41

events until the desired gene has been assembled. In case of longer DNA fragments, afinal PCR step is often included to ensure the amplification or stitching of the shorter
fragments. Another example in this category takes advantage of a combination of several enzymes, such as T5 exonuclease, Taq ligase, and Phusion DNA polymerase toassemble DNA fragments up to several hundred kilobases in vitro83. In this approach, T5
exonuclease first digests the 5’ end of overlapping DNA duplexes to generate
single-stranded, complementary fragments. These fragments then serve as primers for
Phusion DNA polymerase which fills in the empty overlapping areas. Lastly, nicks in theassembled sequence are sealed with a thermostable Taq ligase.
Application of a thermostable enzymes in ligation-mediated assembly techniques
allows for high-temperature annealing and ligation steps that improve the hybridization
stringency and the likelihood of obtaining correct final sequences, which may offer anadvantage over PCA. However, this method becomes in efficient as the number ofoligonucleotides increases; limiting its usefulness to genes shorter than 2 kb84. Furthermore, the use of standard-purity oligonucleotides can introduce point deletions resulting from incorporation of incorrectly synthesized oligonucleotides. This can be avoided
by using gel-purified oligonucleotides, high-quality synthetic oligonucleotides, or by
performing side-directed mutagenesis to correct errors after assembly, cloning, andsequencing.

3.C. Recombination-Mediated AssemblyThis category of assembly methods, performed both in vitro and in vivo, is most suitable
for the fabrication of very large DNA molecules and combines the assembly step withdownstream cloning. The principle is founded on assembling small DNA fragments
containing specific sequences with homology to other fragments within the designed
gene and a plasmid vector. For example, in Sequence- & Ligation-Independent Cloning(SLIC), which is a method of in vitro homologous recombination and single-strand
annealing in one reaction, a T4 DNA polymerase is employed to assemble up to 10 genefragments via simultaneous incorporation into a plasmid vector85. Using PCR extension
to introduce flanking regions of sequence homology, synthetic oligonucleotides areprepared for SLIC; this facilitates recombination of fragments without any sequence
restrictions or the introduction of restriction enzyme sites that will produce permanent
seams. The exonuclease activity of T4 DNA polymerase generates single-stranded DNA
overhangs in the insert and vector sequences. Homology regions are annealed in vitro
and undergo gap repair after transformation into E. coli.
Assembly of mega base-length DNA, however, requires in vivo recombination assembly, which is usually carried out in Bacillus subtilis, E. coli, or yeast. Using a domino
method, for example, 4–6-kb DNA fragments termed “dominos”, are synthesized suchthat the ends of the DNA contains regions of homology on one end to the B. subtilis
genome and on the other end to another domino fragment. Once these pieces areconstructed in conventional E. coli plasmids carrying antibiotic resistance genes, the42

entire designed DNA sequence can then be assembled in the B. subtilis genome using
multiple rounds of transformation and selection86. Using high efficiency recombination
enzymes, such as lambda Red in bacteria as well as employing the intrinsic highefficiency system of homologous recombination in yeast and its ability to replicate large
DNA molecules, a variety of recombination-based methods have been developed.
In vivo homologous recombination in yeast facilitates the assembly of very long
synthetic DNA sequences either from “bricks” of 500-1,000 bp produced through invitro methods, or directly from short oligonucleotides. Recently, the creative work ofGibson and colleagues has demonstrated that the yeast Saccharomyces cerevisiae cantake up and assemble at least 38 overlapping single-stranded oligonucleotides and alinear double-stranded vector in one transformation event. These oligonucleotides canoverlap by as few as 20 bp and can be as long as 200 nucleotides in length to produce
megabase-sized synthetic DNA molecules32, 83. This method employs three enzymatic
activities in a single, isothermal reaction: (1) 5´ exonuclease activity chews back the 5´
end of DNA fragments and exposes the complementary sequence for annealing, (2) the
3´ extension activity of a DNA polymerase fills in the gaps on the annealed regions, andfinally (3) the DNA ligase activity seals the nick and covalently links the DNA fragments
together for final placement of the synthetic gene(s) in the destination vector. Integration of multiple methods with Gibson Assembly is now enabling the modular construction of large, multigene plasmids for mammalian cell experiments, which is widely usedin synthetic biology87.

GenScript's GenBrick™ synthesis service delivers 8-13 kb full-length,
synthetic DNA fragments with 100% guaranteed sequence accuracy. Have asequence longer than that? Just submit your sequence to us and we’ll delivera customized quote and timeline, all based on your research needs!

43Main Assembly Design Platforms

Platform

Features

J5

Designs flanking homology sequences or overhangs for each assembly piece
Enables scar-less multipart and combinatorial DNA construction
Applicable to broad classes of DNA construction methodologies
Compatible with many assembly protocols
Optimizes for cost and parts re-use
Requires carefully-edited raw data
Graphical user interface
Licensing restrictions

Raven

Designs oligonucleotide sequences to prepare parts for assembly
Generates optimized assembly plans
Compatible with most popular assembly protocols
Optimizes for cost and parts re-use
Requires carefully-edited raw data
Graphical user interface
Licensing restrictions

Pydna

Designs DNA constructs and assembly plans
Useful for complex or combinatorial DNA molecule construction
Compatible with Gibson and recombination-based assembly methods
Free and open source
Flexible and extensible
Bottom-up approach allows gradual building and testing for complex projects
Requires basic knowledge of Python coding language

GibthonDesigns primers for Gibson AssemblyWeb-based and interactive
Free and open source

BioPartsBuilder

Designs DNA constructs and assembly plans
Compatible with combinatorial assembly methods
Web-based and interactive
Free and open source

44

4

Sequence Verification & Error Correction

Once the desired synthetic gene has been assembled, it needs to be verified to ensure
the final sequence matches the one originally designed. Due to inherent potential for
error in each step of the gene synthesis process, the assembled DNA product contains
a mixture of perfect and imperfect sequences. Internal insertions, deletions, andtruncated fragments are the result of inefficient coupling, unwanted DMT cleavage,
incomplete capping, or deprotection during the cyclical synthesis process. Improper
annealing during oligo assembly can also introduce heterogeneity in the final pool ofsynthetic gene products. Also sequences harboring mutations must be identified andremoved from the pool or corrected. To screen synthetic products and correct potential
errors, several methods have been developed:
Oligonucleotide and/or gene fragment purification: Since the quality and purityof the oligonucleotides used in the process of chemical gene synthesis have thepotential for errors, many gene synthesis protocols rely on purification of full-length
oligonucleotides using PAGE or agarose gel electrophoresis prior to assembly. A
similar approach is also used for selecting correct gene fragments before assembly.
This approach, though helpful, is time- and labor-intensive and does not identify orcorrect for substitutions, small insertions, and deletions.
Cloning and sequencing: Typically, newly synthesized sequences are cloned intoa plasmid vector and subjected to Sanger sequencing. Sequencing primers thatbind to vector regions flanking the gene insert ensure correct sequencing of theends of the synthesized gene insert. Further, plasmid DNA can be clonally amplified
to create a homogeneous pool of DNA with the correct sequence. This approach is
specifically useful for larger DNA molecules in which segments of the gene or
genome, called synthons, are first cloned and sequenced before the whole gene/genome is assembled86. This approach can be expensive, time consuming, and difficult
to automate.
Reading frame selection: For synthetic protein-coding sequences, functional
selection can be used to prescreen and enrich mutation-deficient sequences. Sincemany of the accumulated errors are single point mutations leading to frame shift andpremature translation termination, the synthetic genes can be cloned into aframe-shift selection vector that will express a fusion reporter protein as a selectable
marker, such as antibiotic resistance or fluorescence markers. Selection is, hence,
based on the survival of intact genes transformed into bacteria and plated on selective media.
Mismatch binding and cleavage: This approach takes advantage of mismatch
repair endonucleases, which bind to error-containing sequences and affect theirelectrophoretic motility. Error-free DNA fragments migrate faster on an electrophoresis gel, and can be eluted and further assembled. The efficiency of this approach is,
however, dependent on the capability of the enzyme. For example, the widely-used
E. coli MutHLS is not very effective for substitutions other than G-T, A-C, G-G, A-A.
45

Site-directed mutagenesis: Following gene assembly or sequencing, primers
containing the targeted correction can be used to directly correct errors throughsite-specific mutation. These primers can be the original ones designed for thesynthesis and assembly of the gene(s) or newly synthesized. This approach,although dependent on one or two extra assembly steps, is very efficient in correcting errors.
NGS-based error correction: Advances in Next Generation Sequencing (NGS) haverecently been employed to screen and then select for perfect sequences at both theoligonucleotide and gene level. Using array sequencing as is, or integrated with tags
and biomarkers, all variations of this approach help expedite screening and selection of correctly-synthesized DNA molecules. While these techniques still need
improvements in sequencing and length-accommodation, they are promising inthat they enable simultaneous correction without the need to separate each geneassembly into individual reactions79.
Since all error detection and correction strategies are time-consuming and costly,
especially for long or complex sequences, efforts continue to improve the accuracy of
initial oligonucleotide synthesis and assembly steps. Recent strategies that rely on
synthesizing and assembling fragments in a single reaction is one example in whichhigher percentage of correct and ready-to-use synthetic products of very long sequences are obtained.

GenScript’s custom gene synthesis platform enables mutation-free DNA
synthesis for genes of any length and any complexity. Sequencechromatograms are delivered with all gene synthesis orders, and 100%
sequence accuracy is always guaranteed! Utilize GenScript’s Gene Synthesis
Services and get straight to the research that matters!

5

Downstream Application Preparation

The correct final product of the chemical synthesis process can be used directly in a few
downstream applications. However, for the majority of applications, synthetic genes
and genomes need to be cloned and propagated. Development of recent synthesis
techniques in which assembly and cloning are combined can expedite the preparationof the product for downstream applications, but sub-cloning and propagation may still
be required for alternative applications.

46

5.A. Cloning
Depending on the specific downstream application, synthetic genes can be cloned into
plasmid vectors for transfection or electroporation into cells or viral vectors (such as
adenoviral, retroviral, or lentiviral vectors) for transduction into cells or live animals.
Cloning methods can also vary from traditional, multi-step techniques to more recent,one-step methods. Currently, main cloning methods include restriction enzyme
cloning, TOPO® cloning, Gateway® recombination cloning, isothermal assembly
reaction or Gibson Assembly®, and type II S assembly, such as Golden Gate. The choiceof cloning strategy depends on the synthetic gene and its intended application goal.
Alternatively, specific features and requirements of each cloning method dictate specific required modifications in the synthetic genes before cloning can occur.

Cloning Vectors
Type

InsertSize (kb)

Host

Delivery
MethodFeatures

Bacterial
plasmid0.1-15

E. coliTransformation

Circular; Double stranded; Smallsize; Single restriction site;
Contains suitable markers forselection; Multiple cloning sites

Bacteriophage10-300E. coliTransduction
Transduction orLinear; Single stranded; Moreefficient than plasmids

Cosmids

35-45

E. colitransformation

Behaves as both plasmid andphage vectors; Hightransformation efficiency

BAC

100-300

E. coliElectroporation

Linear or circular; Suitable forlarge eukaryotic genomes andgenome analysis

YAC

Human
Artificial
Chromosome

47200-2000

S. cerevisiae

Transformation

Linear or circular; Low
transformation efficiency; Suitablefor very large eukaryotic genomes
and mapping complex eukaryotic
chromosomes>2000

Mammalian &human cells

Microcell-mediated chromosome transfer

Linear or circular; Suitable for verylarge eukaryotic genomes, genedelivery and insertionalmutagenesis

Main Cloning Strategies
Strategy

Methodology

FeaturesEﬃciency

Classic, cheap and affordable
Convenient for subcloning
Generates blunt or sticky ends
Restriction
DigestCloning

Restriction
enzymedigestion of

Can clone only one fragment per reaction
Variableinsert andCompatible with many vectory types
Easy to design

vectorLeaves a scar or seamCan be directional or not
Sequence limitations
Suitable for sequencing
Can clone only one fragment per reaction
Amenable to high-throughput

Hybridization
T/A
Cloningof single
base

Easy to design
Variableoverhangs inLeaves a single base insertion scar
Not directional

insert andMulti-fragment cloning not straightforward

vectorNot compatible with many vectors
Time-consuming preparation and costly
Requires an entry vector
Easy and time-saving strategy for subcloning
Can clone several fragments per reaction

Gateway
Cloning

Leaves an "att" site scar

Recombining"att" sites in
insert anddonor vector

Can be directional or not~95%

Fast and accurate
Compatible with many vectory types
Relatively easy to design
Requires an entry vector
Time-consuming initial set up

48

Strategy

Methodology

EﬃciencyFeatures
Can clone several fragments per reaction withhigh level of control
Leaves no scars or seams
Directional
Compatible with many vectory types

GibsonIsothermal

Assembly &assembly of

Cloning

homologus

No sequence limitations~90%
One-step assembly and cloning

overlapping

Easy for multiple DNA manipulations, such as
mutagenesis

sequences

Fast
Difficult to design
High cost of synthesizing overlapping primers
LigationIndependent
Assembly &CloningCompatible with many vectory types
Complementary
overhangs
between insert
and vector

Low cost~95%
Fast
Limitations on sequence modifications
Can clone several fragments per reaction

Golden Gate
Assembly &Cloning

Leaves no scars or seams

Restriction
enzyme (Type II

Directional

S-dependent)
digestion~95%

Re-ligation prone
Suitable for generating combinatorial libraryLess sequence-independent
Difficult to design49Integration of advanced gene synthesis and cloning strategies are now providing scientists with “off-the-shelf” products to facilitate and expedite research. These products
contain the desired gene sequence(s) already cloned in a variety of vectors for directordering and application in an experiment. Products containing the Open Reading
Frame (ORF) of a gene in wild or mutant types, offered as ORF Clones, which aredesigned and ordered through virtual molecular cloning platforms, are suchready-to-transfect or -express products. Pre-fabricated gene fragments corresponding
to functional or regulatory elements of a gene and its ORF can also be ordered for directuse in an application or cloning for future applications.

5.B. Propagation
Synthetic genes and genomes can be propagated through the amplification of theplasmid they are cloned in. This procedure, however, may be challenging for several
reasons: some “low copy number” plasmids simply don’t amplify well in commonly-used bacterial propagation hosts; long genes are often diffcult to maintain and propagate because of the energetic burden on the host cell; some genes may alter the physiology of their hosts, creating aberrant culture temperature requirements or other conditions that may be difficult to identify and accommodate; and genes may be toxic or
unstable in certain host cells. One solution is to screen a number of cell lines to find thebest host for propagation. While this strategy can be time consuming, but it may benecessary for certain ii sequences.

GenScript's Express Plasmid Prep Services, in both research andtransfection grade packages, can accommodate any size and quantity of DNA
preparations to help you achieve highly efficient cell transfection for DNA
vaccine development, antibody production, and other preclinical research
projects.

50

Chapter Four
Perspectives

Biosafety
Throughout the history of genetics research, numerous regulatory boards haveaddressed the safety concerns of researchers, citizens, and political leaders regarding
genetic technologies. The potential for genetic technologies to be used for harm has
always been a major point of discussion with each new scientific advancement. In
1974, the National Institutes of Health formed Recombinant DNA Advisory Committee(RAC) to review recombinant DNA safety protocols and address public misgivings
about newly acquired scientific and technological powers to manipulate genetic
materials. Although gene synthesis allows revolutionary flexibility for researchers, it
poses no qualitatively different risks than those that have already been regulated foralmost half a century since the advent of recombinant DNA technology. In 2009 theInternational Gene Synthesis Consortium (IGSC) was established to work closely withthe research community and the Presidential Commission on the Study of BioethicalIssues to evaluate and manage biosafety risks. The IGSC has developed industry-wide
protocols to screen all synthetic gene orders against databases maintained by national
and private agencies to identify regulated pathogen sequences and other potentially
dangerous sequences in order to prevent their misuse.

GenScript is proud to be a founding member of the International Gene
Synthesis Consortium (IGSC), whose member companies represent morethan 90% of the worldwide gene synthesis capacity.

FutureGene synthesis technology has revolutionized both our understanding of how DNA
functions as the blueprint of life and our ability to manipulate DNA for experimental,
medical, and industrial purposes. While the capabilities and speed of gene synthesis
have steadily increased, its cost has dropped from $10 per bp to $0.09 per bp andbelow over the last decade; in parallel with advances in DNA sequencing andchip-based bioassays. Further improvements in automation, assembly, error correction,
high-throughput platforms, and cost-effectiveness will further allow gene synthesis to52

be not only a valuable, but also an indispensable tool for a widening range of scientificdisciplines and economically important applications. The energy and agriculture industries will increasingly rely upon gene synthesis to solve problems related to environmental protection and food supplies. DNA-based vaccines and bioengineered antibodies will become more widely used in medicine. Designer organisms housing customized metabolic pathways will become commonplace. Tinkering with the cellular circuitry to modulate biosynthetics, including synthetic sensors and regulators, will continue
to be a focus of synthetic biology research. The potential environmental and social
impacts of integrating synthetic genes into endogenous systems will kindle ongoing
discussions among the research community, biotech industry, governmental andinternational regulatory bodies. Without a doubt, the coming decade will present
exciting opportunities for gene synthesis to continue to fuel innovation in life sciences.

53

Chapter Five
Resources

ToolsTo help facilitate the design and application of your synthetic genes, GenScript hasdeveloped a comprehensive set of powerful tools. The following introduction familiarizes you with the scope of each tool before you are ready to use them online through our
secure and encrypted system at https://www.genscript.com/gene_technical_resources.html.

+ GenSmart Design

Helps you integrate all you need to design a DNA
construct into 1 of the 3 simple, drag-and-drop
operations: insertion, fusion and replacement. This
smart construct design interface requires no
installation and can automatically annotate yourDNA sequence and draw plasmid map.

+ GenSmart Codon Optimization

Enables you to optimize the design of wild type or
recombinant gene sequences towards higher
expression in prokaryotic and mammalian expression systems. Developed based on population
genetics and immunology theories, this free tool
takes more than 200 factors involved in gene
expression into consideration to maximize thechance of obtaining a functional and active protein.

+ Rare Codon Analysis Tool

Determines the codon usage frequency anddistribution of your target sequence. It can help you
decide if your sequence needs to be optimized forheterologous gene expression.

+ Restriction Site Map

Analyzes your target sequence for restriction sites
and displays where restriction enzymes will cut.

+ Gene Mutagenesis

Facilitates the design of point mutations in yourtarget sequence. Simply submit the full sequence,desired expression system, and location of eachmutation to obtain the right mutated sequence.

+ Oligo Calculation Tool

Calculates melting temperature (Tm), molecularweight (MW), and millimolar extinction coefficient(OD/μmol) and μg/OD of oligonucleotides.

+ siRNA Target Finder

Identifies proper sequences within your sequencethat would be efficiently targeted by synthetic
sequences for gene silencing in your target
organism or organ.

Analysis Tool

Designer55

+ siRNA Construct Builder

Helps you build small hairpin inserts for use as
siRNA expression vectors.

+ ORF Clone Vector List

Helps you find the right expression vector for
the ORF clone of your choice by consideringfactors such as the expression host, promoter,bacterial selection, copy number, or epitopetag.

+ Sequence Manipulation

Provides a collection of JavaScript programsfor generating, formatting, and analyzing short
DNA and protein sequences.

Suite

Design Considerations
To help you design functional synthetic genes and solve problems related to genesynthesis and application, consider the following guide in conjunction with ourFrequently Asked Questions page at our Gene Technical Resource Center: https://www.genscript.com/gene_technical_resources.html. Our Ph.D.-level technical accountmanagers are also available to answer your questions 24/7.

General Considerations
Advanced and thorough preparation is key in obtaining a functional synthetic
nucleic acid product. Answering the following questions helps to prepare for yourproject:
What is your specific scientific question?
How does a synthetic gene help answer your question?
Are you looking at the function of a gene or how it is regulated?
Do you need the complete gene sequence or part(s) of it?
Is it necessary to include regulatory elements of your gene of interest in the synthetic
construct?
Do you need the wild-type or mutated form of the gene?
Do you need to have more than one gene sequence in your construct?
Do you need to have the expressed, purified product of the synthetic gene(s) orassessing its cellular function suffice?
Do you need to include reporter tags or molecules in your construct, and if so, where
does it need to be placed to avoid interference with gene function?
What biological system, prokaryotic or eukaryotic, can provide you with the mostfunctional product?
56

What synthesis and assembly methods are the most appropriate to employ for
obtaining a functional synthetic product?
What are the requirements for your choice of synthesis and assembly methods toensure proper oligonucleotide design for a successful process?

Oligonucleotide Design
Designing oligonucleotides for the synthesis of long DNA sequences or genomes
can be a time-consuming, difficult, and confusing process. Make sure to know thedetails of your target sequence(s) and follow the specific requirements of your selected assembly method and cloning strategy.
Particular aspects of the desired sequence(s) can interfere with gene synthesis andassembly. Pay attention to codon usage, GC content, restriction enzyme sites, andpotential for secondary structures within your desired sequence(s). Most of theseproperties can be eliminated by juggling codon usage in a sequence.
DNA repeats in the target sequence may occur in many forms including direct, inverted, palindromic, or tandem repeats. Presence of repetitive DNA sequences can leadto large deletions in homology-directed assembly reactions. In such cases, it is best
to use restriction enzyme-based assembly strategies to avoid sequence-specificannealing reactions. Repeated sequences may also pose challenges for correct
annealing or ligation of oligonucleotides during assembly.
GC content determines the stability of DNA strands and thus the melting temperature. All in vitro assembly methods, both ligase and polymerase based, rely upon themelting and annealing of oligonucleotides and as a result require that oligonucleotides have equal melting temperatures.
Improper hybridization or intramolecular binding, such as hairpin structure formation, can be avoided through careful design of oligonucleotides, including theoverhangs, or primers that were used for assembly.
To avoid introducing special flanking sequences in recombination-based assembly
methods, introduce 15 bp with sequence homology to the linearized vector through
PCR extension of the gene insert. This will facilitate homologous recombination
without adding unwanted bases.
Since cellular intermediates are a component of gene fabrication strategies,
presence of toxic genes can interfere with gene fabrication. Make sure to check theirpresence in your target sequence(s). If they exist, avoid them by omitting their startcodon or introduce a frameshift mutation to disrupt their activity. Once the syntheticgene(s) are fabricated, you can repair the mutation through site-directed mutagenesis.
While synthesis platforms utilizing phosphoramidite chemistry have very low error
rates, errors do accumulate as strand length increases; therefore, it is recommended
to use oligonucleotides with lengths of 40-200 bp. The optimal oligo length depends
upon the assembly method that will be used, the complexity of the sequence, andyour preferences.
57

As a general rule, shorter oligonucleotides may have a lower error rate, but will be
more expensive to synthesize because more overlaps will be required. Longer
overlaps increase the likelihood of correct assembly by decreasing the rate ofnonspecific annealing.
Oligonucleotides should be adjusted for even lengths and equal melting temperatures. Pay attention to parameters of oligonucleotide design tools if you choose to
use them. Each tool has its own default parameters; for example, Gene Design uses
default settings of 60 bp oligonucleotides with 20 bp overlaps; values which typically work well with yeast and mammalian sequences which have about 40% GC
content.

GenScript’s OptimumGene™ is the industry-leading gene optimization
service that takes multiple variables influencing gene expression into account inorder to provide you with the ideal sequence for your research needs.

Plasmid Prep & Cloning
Depending on your research needs, the quality of plasmid obtained from your genesynthesis project may need varying levels of quality control. Key factors are
A260/280, residual RNA, endotoxin levels, and supercoiling percentages.
It is essential for successful cloning that the plasmid purity is high. DNA absorbanceas measured by a spectrophotometer can be used to determine the purity after
plasmid purification: DNA, proteins, and solvents absorb at 260 nm, 280 nm, and 230
nm, respectively. The ratio of absorbance at 260 and 280 nm is used to assess thepurity of nucleic acids. An OD260/280 ratio of >1.8 and an OD260/230 ratio of 2-2.2 areindicative of pure for DNA samples.
Residual RNA in a plasmid prep can interfere with downstream applications. It is
recommended to remove RNA with DNase-free RNase or use a Sephacryl S-300
column.
Supercoiled plasmids have a better transformation efficiency than plasmids that are
relaxed. Relaxed plasmids are transformed with 25% less effciency than supercoiled
plasmids. Thus, higher levels of supercoiling in a plasmid prep results in a higher
subsequent transformation effciency.
Endotoxins are molecules that are comprised of a lipid and a polysaccharide. They
are can be found within a bacterial cell and are released when the bacterial cell wallis destroyed (during the plasmid prep procedure). The removal and protection
against contamination by these lipids are key in certain applications to avoid
contaminating experiments and to avoid toxicity. Here are some applications that
require lower endotoxin levels:
58

Transfection and co-transfection of mammalian cells
Antibody production
Gene vaccine and gene therapy studies
Protein production
Animal studies

Bundling Gene Synthesis Services from GenScript with Express Cloning
Services offers you free access to over 150 popular vectors for cloning yourperfectly-synthesized sequence into mammalian, bacterial, yeast, or insect
expression systems.

Storage & HandlingAfter receiving the vials containing plasmids, make sure to store them at 4°C for
short-term and frequent use, or at -20°C for long-term storage.
Keep vials sealed until ready to use.
Prior to use, centrifuge the vial at 6,000 x g for 1 minute at 4°C. Open the vial and add
20 μl of sterilized water to dissolve the DNA. Close the lid and vortex the vial for 1
minute. If necessary, heat the solution at 50°C for 15 minutes to dissolve the DNA.
Transformation of the plasmid DNA can be performed directly after completion of thesteps above. Verify the plasmid sequence after each subcloning and transformation
step, unless you obtained your plasmid from a trusted vendor who ensuressequence verification.
If you received your plasmid in glycerol, avoid repeated thawing and re-freezing of
the glycerol stocks since it may reduce bacterial viability.

By taking advantage of GenScript’s VectorArk Storage and VectorArk
Services, you can securely store your vectors and customized plasmids for
future cloning projects for free. Leave the worry of losing your precious clone
behind!

59

References

1. Michelson, AM. and Todd, AR. Nucleotides part XXXII. Synthesis of a dithymidine
dinucleotide containing a 3:5 -internucleotidic linkage. Journal of the Chemical Society(Resumed). 1955; 0: 2632–2638.
2. Agarwal, KL. et al. Total synthesis of the gene for an alanine transfer ribonucleic acidfrom yeast. Nature. 1997; 227: 27–34.
3. Smith HO, et al. Generating a synthetic genome by whole genome assembly: phiX174
bacteriophage from synthetic oligonucleotides. Proc Natl Acad Sci U S A. 2003;
100(26):15440-5.
4. Tian J. et al. Advancing high-throughput gene synthesis technology. Mol Biosyst.
2009; 5(7):714-22.
5. Rothstein RJ. One-step gene disruption in yeast. Methods Enzymol. 1983;
101:202-11.
6. Kimmel AR. And Faix J. Generation of multiple knockout mutants using the Cre-loxP
system. Methods Mol Biol. 2006; 346:187-99.
7. Gaj, T., et al. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.
Trends Biotechnol. 2013; 31:397–405.
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64

Notes65

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